Monitoring recombinase polymerase amplification mixtures

ABSTRACT

A process includes providing a mixture that includes a recombinase, a single-strand binding protein, and one or more oligonucleotides; and detecting particles in the reaction mixture.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation and claims priority to U.S. patentapplication Ser. No. 14/183,113, filed Feb. 18, 2014, now U.S. Pat. No.9,157,127, which is a continuation and claims priority to U.S. patentapplication Ser. No. 13/441,411, filed Apr. 6, 2012, now U.S. Pat. No.8,809,021, which claims priority to U.S. Patent Application Ser. No.61/472,929, filed Apr. 7, 2011, the entire contents of which areincorporated by reference.

TECHNICAL FIELD

This disclosure relates to methods and compositions for nucleic aciddetection, amplification, and quantitation.

BACKGROUND

Certain isothermal amplification methods are able to amplify template(target) nucleic acid in a specific manner from trace levels to veryhigh and detectable levels within a matter of minutes. Such isothermalmethods, e.g., Recombinase Polymerase Amplification (RPA), can broadenthe application of nucleic acid based diagnostics into emerging areassuch as point-of-care testing, and field and consumer testing. Theisothermal nature and broad temperature range of the technologies canallow users to avoid the use of complex power-demanding instrumentation.

SUMMARY

This disclosure is based, at least in part, on the observation ofparticles within RPA mixtures. In some embodiments, these particles caninclude nucleic acids (e.g., oligonucleotides) and/or protein componentsof the RPA reaction. This discovery provides for new monitoring anddetection methods relating to RPA.

In one aspect, this disclosure features processes that include: (a)providing a mixture that includes one or more of (e.g., two or more of,or all of) a recombinase, a single-stranded DNA binding protein, and oneor more nucleic acids (e.g., oligonucleotides) (in any combination); and(b) detecting particles in the reaction mixture. In some embodiments,the mixture includes a crowding agent, e.g., one or more of polyethyleneglycol (e.g., PEG1450, PEG3000, PEG8000, PEG10000, PEG14000, PEG15000,PEG20000, PEG250000, PEG30000, PEG35000, PEG40000, PEG compound withmolecular weight between 15,000 and 20,000 daltons, or combinationsthereof), polyvinyl alcohol, dextran and ficoll. In some embodiments,the crowding agent is present in the reaction mixture at a concentrationbetween 1 to 12% by weight or by volume of the reaction mixture, e.g.,between any two concentration values selected from 1.0%, 1.5%, 2.0%,2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%,8.5%, 9.0%, 9.5%, 10.0%, 10.5%, 11.0%, 11.5%, and 12.0%.

In some embodiments of all aspects, the recombinase includes a RecA orUvsX recombinase. In some embodiments of all aspects, thesingle-stranded DNA binding protein includes a prokaryotic SSB proteinor a gp32 protein. In some embodiments of all aspects, at least one ofthe one or more nucleic acids (e.g., oligonucleotides) includes adetectable label.

In some embodiments of all aspects, the particles include one or more(e.g., two or more, or all) of a recombinase, a single stranded bindingprotein, and at least one of the one or more nucleic acids (in anycombination). In some embodiments of all aspects, the reaction mixtureincludes a recombinase, a single-stranded binding protein, a polymerase,dNTPs, ATP, a primer, and a template nucleic acid.

In some embodiments of all aspects, the mixture includes one or more(e.g., two or more, three or more, four or more, five or more, six ormore, seven or more, eight or more, or all) of a recombinase, a DNApolymerase, a single-stranded binding protein, a recombinase loadingprotein, ATP, dNTPs or a mixture of dNTPs and ddNTPs, a reducing agent,creatine kinase, a nuclease (e.g., an exonuclease III or endonucleaseIV), a reverse transcriptase, a nucleic acid probe, a nucleic acidprimer, and a template nucleic acid (in any combination).

In some embodiments of all aspects, the particles include a polymerase,dNTPs, ATP, a primer, and a template nucleic acid. In some embodimentsof all aspects, the particles include a recombinase, a polymerase,dNTPs, ATP, a primer, and a template nucleic acid. In some embodimentsof all aspects, the particles include a recombinase, a single-strandedbinding protein, a polymerase, dNTPs, ATP, a primer, and a templatenucleic acid. In some embodiments of all aspects, the particles includea polymerase, dNTPs, and ATP, and one or more (e.g., two, three, four,five, or six) additional agents selected from the group of a probe, aprimer, a single-stranded binding protein, ddNTPs, a reducing agent,creatine kinase, a nuclease, and a reverse transcriptase. In someembodiments of all aspects, the particles include a recombinase, apolymerase, a reverse transcriptase, dNTPs, ATP, a primer, and atemplate nucleic acid.

In some embodiments of all aspects, the particles are about 0.5-20 μm insize, e.g., between about any two sizes selected from 0.5, 1, 1.5, 2,2.5, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, and 20 μm (e.g., about 1-10 μmin size).

In some embodiments of all aspects, approximately 10 to 5000particles/nL, e.g., between any two numbers of particles selected from10, 20, 50, 100, 200, 500, 1000, 2000, and 5000 particles per nL, aredetected.

In some embodiments of all aspects, detecting particles in the mixtureincludes the use of one or more of microscopy, a microfluidic device,flow cytometry, and a camera. In some embodiments of all aspects, theparticles are detected using charge-coupled detection (CCD).

In another aspect, the disclosure features a process that includes: (a)providing a recombinase polymerase amplification reaction mixture; (b)maintaining the reaction mixture under conditions that allow for theproduction of nucleic acid amplification products in the reactionmixture; and (c) detecting particles associated with the nucleic acidamplification products in the reaction mixture. In some embodiments, thedetecting is performed within 10 minutes (e.g., within 9, 8, 7, 6, 5, 4,3, 2, 1.5, or 1 minute) from when the maintaining begins.

In some embodiments of all aspects, the reaction mixture includes acrowding agent, e.g., one or more of polyethylene glycol (e.g., PEG1450,PEG3000, PEG8000, PEG10000, PEG14000, PEG15000, PEG20000, PEG250000,PEG30000, PEG35000, PEG40000, PEG compound with molecular weight between15,000 and 20,000 daltons, or combinations thereof), polyvinyl alcohol,dextran and ficoll. In some embodiments, the reaction mixture containspolyethylene glycol as a crowding agent (e.g., any of the PEG compoundsdescribed herein or known in the art). In some embodiments, the reactionmixture contains polyvinyl alcohol as a crowding agent. In someembodiments, the crowding agent is present in the reaction mixture at aconcentration between 1 to 12% by weight or by volume of the reactionmixture, e.g., between any two concentration values selected from 1.0%,1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%,7.5%, 8.0%, 8.5%, 9.0%, 9.5%, 10.0%, 10.5%, 11.0%, 11.5%, and 12.0%. Insome embodiments, the crowding agent is present in the reaction mixtureat a concentration that is sufficient to increase the amount ofamplification in the reaction mixture.

In some embodiments of all aspects, the particles include one or more(e.g., two or more or all) of the recombinase, the single strandedbinding protein, and at least one of the one or more nucleic acids (inany combination).

In some embodiments of all aspects, the reaction mixture includes one ormore (e.g., two or more, three or more, four or more, five or more, sixor more, seven or more, eight or more, or all) of a DNA polymerase, arecombinase loading protein, ATP, dNTPs or a mixture of dNTPs andddNTPs, a reducing agent, creatine kinase, a nuclease (e.g., anexonuclease III or endonuclease IV), a single-stranded binding protein,a nucleic acid primer, a nucleic acid probe, reverse transcriptase, anda template nucleic acid (in any combination).

In some embodiments of all aspects, the reaction mixture contains arecombinase, a single-stranded binding protein, and one or moreoligonucleotides. In some embodiments of all aspects, the reactionmixture includes a recombinase, a single-stranded binding protein, apolymerase, dNTPs, ATP, a primer, and a template nucleic acid.

In some embodiments of all aspects, the reaction mixture includes apolymerase, dNTPs, ATP, a primer, and a template nucleic acid. In someembodiments of all aspects, the reaction mixture includes a recombinase,a polymerase, dNTPs, ATP, a primer, and a template nucleic acid. In someembodiments of all aspects, the reaction mixture includes a recombinase,a single-stranded binding protein, a polymerase, dNTPs, ATP, a primer,and a template nucleic acid. In some embodiments of all aspects, thereaction mixture includes a polymerase, dNTPs, and ATP, and one or more(e.g., two, three, four, five, or six) additional agents selected fromthe group of a probe, a primer, a single-stranded binding protein,ddNTPs, a reducing agent, creatine kinase, a nuclease, and a reversetranscriptase. In some embodiments of all aspects, the reaction mixtureincludes a recombinase, a polymerase, a reverse transcriptase, dNTPs,ATP, a primer, and a template nucleic acid. In some embodiments of allaspects, the particles are about 0.5-20 μm in size, e.g., between aboutany two sizes selected from 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9,10, 12, 15, 18, and 20 μm (e.g., about 1-10 μm in size).

In some embodiments of all aspects, approximately 10 to 5000particles/nL, e.g., between any two numbers of particles selected from10, 20, 50, 100, 200, 500, 1000, 2000, and 5000 particles per nL, aredetected.

In some embodiments of all aspects, the detecting includes determining anumber or proportion of particles associated with nucleic acidamplification products in the reaction mixture and, optionally,determining or estimating the concentration of template nucleic acid inthe original mixture thereby. In some embodiments, the detectingincludes detecting single particles associated with two or more distinctnucleic acid amplification products.

In another aspect, the disclosure features compositions that include (a)a first population of particles that includes a first recombinase, afirst single-stranded DNA binding protein, and a first oligonucleotide;and (b) a second population of particles that includes a secondrecombinase, a second single-stranded DNA binding protein, and a secondoligonucleotide, wherein the first and second oligonucleotides aredifferent. In some embodiments, at least one of the first and secondoligonucleotides includes a detectable label. In some embodiments, thefirst and second oligonucleotides include the same or differentdetectable labels. The first and second single-stranded DNA bindingprotein can be the same or different from each other. The first andsecond recombinase can be the same or different from each other.

In some embodiments of all aspects, the particles are about 0.5-20 μm insize, e.g., between about any two sizes selected from 0.5, 1, 1.5, 2,2.5, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, and 20 μm (e.g., about 1-10 μmin size).

In some embodiments of all aspects, approximately 10 to 5000particles/nL, e.g., between any two numbers of particles selected from10, 20, 50, 100, 200, 500, 1000, 2000, and 5000 particles per nL, arepresent in the compositions.

In some embodiments of all aspects, the compositions include a crowdingagent, e.g., one or more of polyethylene glycol (e.g., PEG1450, PEG3000,PEG8000, PEG10000, PEG14000, PEG15000, PEG20000, PEG250000, PEG30000,PEG35000, PEG40000, PEG compound with molecular weight between 15,000and 20,000 daltons, or combinations thereof), polyvinyl alcohol, dextranand ficoll. In some embodiments, the crowding agent is present in thecomposition at a concentration between 1 to 12% by weight or by volumeof the reaction mixture, e.g., between any two concentration valuesselected from 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%,5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, 10.0%, 10.5%,11.0%, 11.5%, and 12.0%.

In some embodiments of all aspects, the compositions further include oneor more (e.g., two or more, three or more, four or more, five or more,six or more, seven or more, eight or more, or all) of a DNA polymerase,a recombinase loading protein, ATP, dNTPs or a mixture of dNTPs andddNTPs, a reducing agent, creatine kinase, a nuclease (e.g., anexonuclease III or endonuclease IV), a nucleic acid probe, and atemplate nucleic acid (in any combination).

In some aspects, the disclosure features compositions that include oneor more oligonucleotides describe herein and variants thereof. In someembodiments, the oligonucleotides can be used as primers and/ordetection probes in methods of nucleic acid amplifications (e.g.,isothermal nucleic acid amplifications such as RPA). Theoligonucleotides described herein can include one or more detectablelabels. Where an oligonucleotide is disclosed as including one or moredetectable labels, alternative labels may be used at the same positionsor at different positions within the oligonucleotide (e.g., at aposition within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 25, or 30 bases 5′ or 3′ of the disclosed position). Insome embodiments, the oligonucleotides can include one or more abasicsite mimics. Where an oligonucleotide includes one or more abasic sitemimics, alternative abasic site mimics may be included at the sameposition or at different positions within the oligonucleotide (e.g., ata position within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 25, or 30 bases 5′ or 3′ of the disclosed position). Insome embodiments, a variant of an oligonucleotide described herein hastwelve or fewer (e.g., eleven or fewer, ten or fewer, nine or fewer,eight or fewer, seven or fewer, six or fewer, five or fewer, four orfewer, three or fewer, two or fewer, or one or fewer) insertions,deletions, substitutions, and/or additions compared to the disclosedoligonucleotide sequence. In some embodiments, a variant of anoligonucleotide described herein has a sequence at least 80% (e.g., 85%,90%, or 95%) identical to the disclosed oligonucleotide sequence.

In some embodiments, the particles are detected using fluorescence fromthe particles.

In certain embodiments, the particles are detected without usingfluorescence from the particles.

In some embodiments, the particles are detected using fluorescence fromthe particles, phase contrast microscopy, luminescent detection,spectral (color) detection, magnetic detection, radioisotopic detection,and/or electrochemical detection. In some embodiments the particles canbe detected using a combination of two of more (e.g., two, three, orfour) of fluorescence from the particles, phase contrast microscopy,luminescent detection, spectral (color) detection, magnetic detection,radioisotopic detection, and electrochemical detection.

In some embodiments, some of the particles are detected usingfluorescence from those particles, and other of the particles aredetected without using fluorescence from these other particles. Forexample, the particles include a first subset of particles and a secondsubset of particles. The first subset of particles is detected usingfluorescence from the first subset of particles, and the second subsetof particles are detected without using fluorescence from the secondsubset of particles (e.g., phase contrast microscopy, luminescentdetection, spectral (color) detection, magnetic detection, radioisotopicdetection, and/or electrochemical detection).

In another aspect, the disclosure features a population of particlesthat includes a recombinase, a single-stranded DNA binding protein, andan oligonucleotide, wherein some of the particles are detected usingfluorescence from those particles, and other of the particles aredetected without using fluorescence from these other particles.

Also provided are kits including a recombinase, a single-stranded DNAbinding protein, and an oligonucleotide for use in any of the methodsdescribed herein. Also provided are kits including any of the particlesor compositions described herein and instructions for performing any ofthe methods described herein.

The processes and compositions disclosed herein can be used for thedetection of nucleic acids, e.g., bacterial nucleic acids, mammaliannucleic acids, viral nucleic acids, fungal nucleic acids, or protozoannucleic acids, and for the diagnosis of disorders or diseases associatedwith such nucleic acids.

As used herein the “size” of a particle refers to the largestcross-sectional dimension of the particle.

As used herein, an “oligonucleotide” refers to a nucleic acid polymercontaining at least 10 (e.g., at least 12, 15, 20, 25, 30, 35, 40, 50,60, 70, 80, 90, or 100) base units. In some embodiments, theoligonucleotide contains a total of less than 1 kb, 900 base units, 800base units, 700 base units, 600 base units, 500 base units, 400 baseunits, 300 base units, 200 base units, or 100 base units. In someembodiments, an oligonucleotide can have 90 or fewer, 80 or fewer, 70 orfewer, 60 or fewer, 50 or fewer, 40 or fewer, 30 or fewer, or 20 orfewer base units. In some embodiments, an oligonucleotide has at least10, 12, 14, 16, 18, or 20 base units.

As used herein, “cytometry” refers to methods and compositions fordetecting, visualizing, and analyzing the properties of particles. Theterm as used herein does not denote the presence of cells. However,methods and compositions used for detecting, visualizing, and analyzingthe properties of cells can be applied to the particles describedherein.

As used herein, an “abasic site mimic” refers to a subunit positionwithin a nucleic acid polymer in which a sugar or modified sugar moiety(e.g., glucose or deoxyglucose) is present, and the 1′ carbon of thesugar or modified sugar moiety is not covalently bonded to a cyclic basestructure (e.g., adenine, guanine, cytosine, thymine, uracil, ormodified versions thereof). In some embodiments, the 1′ carbon of thesugar or modified sugar moiety is covalently bonded to a hydrogen (e.g.,tetrahydrofuran). In some embodiments, the 1′ carbon of the sugar ormodified sugar moiety is covalently bonded to another carbon that is notpresent in a cyclic base structure. In some embodiments, the 1′ carbonof the sugar or modified sugar moiety is covalently bonded to anon-cyclic linker structure. In some embodiments, an abasic site mimicis recognized by an enzyme which processes and modifies abasic sitemimics due to structural similarity to an abasic site (i.e. lack of abulky base group attached to the sugar).

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,suitable methods and materials are described below. All publications,patent applications, patents, and other references mentioned herein areincorporated by reference in their entirety. In case of conflict, thepresent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and notintended to be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims

BRIEF DESCRIPTION OF DRAWINGS

The patent or application file contains at least one drawing executed incolor (FIGS. 1-13). Copies of this patent or patent applicationpublication with color drawings will be provided by the Office uponrequest and payment of the necessary fee.

FIG. 1 shows micrographs depicting a single field of a mixture includingparticles. The scale bar indicates 100 μm. (A), differentialinterference contrast (DIC). (B), fluorescence. (C), merge.

FIG. 2 shows micrographs depicting a single field of a mixture includingparticles and a template nucleic acid. The scale bar indicates 100 μm.(A), DIC. (B), fluorescence. (C), merge.

FIG. 3 shows fluorescence micrographs (A-H) depicting mixtures includingthe indicated concentrations of polyethylene glycol (PEG).

FIG. 4 shows micrographs depicting mixtures including particles. (A) and(B) are a standard mixture. (C) and (D) are the standard mixtureexcluding UvsX. (E) and (F) are the standard mixture excluding gp32.(A), (C), (E), DIC. (B), (D), (F), fluorescence.

FIG. 5 shows micrographs (A-H) depicting mixtures including particles.(A) and (B) are the standard mixture as in FIGS. 4 (A) and (B), butexcluding UvsY. (C) and (D) are the standard mixture excludingpolymerase. (E) and (F) are the standard mixture excluding creatinekinase. (G) and (H) are the standard mixture excluding exonuclease III.(A), (C), (E), (G), DIC. (B), (D), (F), (H), fluorescence.

FIG. 6 shows sets of micrographs (A-C) depicting mixtures. (A), twofields showing complete mixture. (B), two fields showing completemixture excluding gp32 and UvsY. (C), two fields showing completemixture excluding gp32, UvsY, and Emix (50 mM Phosphocreatine, 2.5 mMATP). For each set: top, DIC; bottom, fluorescence.

FIG. 7 shows micrographs (A-F) depicting a mixture including particlesprepared with two labeled oligonucleotides. (A), Texas red fluorescence.(B), merge DIC and Texas red. (C), FAM fluorescence. (D), merge DIC andFAM. (E), DIC. (F), merge Texas red and FAM.

FIG. 8 shows micrographs (A-F) depicting a mixture including two sets ofparticles with two labeled oligonucleotides prepared independently andthen mixed. (A), Texas red fluorescence. (B), merge DIC and Texas red.(C), FAM fluorescence. (D), merge DIC and FAM. (E), DIC. (F), mergeTexas red and FAM.

FIG. 9 is a time course of micrographs depicting particles during anamplification reaction.

FIG. 10 is a time course of micrographs depicting particles during anamplification reaction.

FIG. 11 is a time course of micrographs depicting particles during anamplification reaction, visualized by DIC/FAM and DIC/Texas Red.

FIG. 12 shows sets of micrographs (A-D) depicting mixtures includingparticles at 20× magnification. (A), mixture including T6 H66S UvsX andUvsY. (B), mixture including T6 H66S UvsX without UvsY. (C), mixtureincluding T6 UvsX and UvsY. (D), mixture including T6 UvsX without UvsY.For each set: top, DIC; bottom, fluorescence.

FIG. 13 shows sets of micrographs (A-D) depicting mixtures includingparticles at 40× magnification. (A), mixture including T6 H66S UvsX andUvsY. (B), mixture including T6 H66S UvsX without UvsY. (C), mixtureincluding T6 UvsX and UvsY. (D), mixture including T6 UvsX without UvsY.For each set: top, DIC; bottom, fluorescence.

FIG. 14 shows line graphs (A-B) depicting amplification reactions inmixtures including T6 H66S UvsX and UvsY (std UvsX+UvsY), T6 H66S UvsXwithout UvsY (std UvsX−UvsY), T6 UvsX and UvsY (T6 UvsX+UvsY), and T6UvsX without UvsY (T6 UvsX−UvsY). (A), 500 copies template. (B), 50copies of template.

FIG. 15 is a line graph depicting amplification reactions in mixturesincluding T6 H66S UvsX and UvsY (std UvsX+UvsY), T6 H66S UvsX withoutUvsY (std UvsX−UvsY), T6 UvsX and UvsY (T6 UvsX+UvsY), and T6 UvsXwithout UvsY (T6 UvsX−UvsY).

FIG. 16 shows line graphs (A-B) depicting amplification reactions inmixtures including T6 H66S UvsX and UvsY (std UvsX+UvsY), T6 H66S UvsXwithout UvsY (std UvsX−UvsY), T6 UvsX and UvsY (T6 UvsX+UvsY), and T6UvsX without UvsY (T6 UvsX−UvsY).

DETAILED DESCRIPTION

On microscopic observation, structures with the appearance of particleswere observed within RPA mixtures. During the progress of the RPAnucleic acid amplification reaction, the particles are associated withloci of active amplification.

The particles observed were typically in the range of 1-10 μm in size,and were present at approximately 100-500 particles/nL. The particleswere found to contain oligonucleotides present in the mixtures.Formation of the particles did not require the presence of magnesium.However, particles formed in the absence of a recombinase or asingle-stranded DNA binding protein had an altered morphology. Formationof the particles in the absence of other agents, such as recombinaseloading protein, DNA polymerase, creatine kinase, or exonucleases, didnot significantly affect particle morphology. Additionally, particleformation was more efficient in the presence of crowding agents.

The particles were observed to be relatively stable in solution.Separate populations of particles could be mixed and remain distinct fora period of time following mixing.

Recombinase Polymerase Amplification

RPA is a method for amplification (e.g., isothermal amplification) ofnucleic acids. In general, in a first step of RPA a recombinase iscontacted with a first and a second nucleic acid primer to form firstand second nucleoprotein primers. In general, in a second step the firstand second nucleoprotein primers are contacted to a double strandedtemplate nucleic acid to form a first double stranded structure at afirst portion of the first strand of the template nucleic acid, and asecond double stranded structure at a second portion of the secondstrand of the template nucleic acid, such that the 3′ ends of the firstnucleic acid primer and the second nucleic acid primer are orientedtowards each other on a given DNA molecule. In general, in a third stepthe 3′ end of the first and the second nucleoprotein primers areextended by DNA polymerases to generate first and second double strandednucleic acids, and first and second displaced strands of nucleic acid.Generally, the second and third steps can be repeated until a desireddegree of amplification is reached.

As described herein, RPA employs enzymes, known as recombinases, thatare capable of pairing oligonucleotide primers with homologous sequencesin template double-stranded DNA. In this way, DNA synthesis is directedto defined points in a template double-stranded DNA. Using two or moresequence-specific (e.g., gene-specific) primers, an exponentialamplification reaction is initiated if the template nucleic acid ispresent. The reaction progresses rapidly and results in specificamplification of a sequence present within the template double-strandedDNA from just a few copies of the template DNA to detectable levels ofthe amplified products within minutes. RPA methods are disclosed, e.g.,in U.S. Pat. No. 7,270,981; U.S. Pat. No. 7,399,590; U.S. Pat. No.7,666,598; U.S. Pat. No. 7,435,561; US 2009/0029421; and WO 2010/141940,all of which are incorporated herein by reference.

RPA reactions contain a blend of proteins and other factors that supportboth the activity of the recombination element of the system, as well asthose which support DNA synthesis from the 3′ ends of oligonucleotidespaired to complementary substrates. In some embodiments, the RPAreaction contains a mixture of a recombinase, a single-stranded bindingprotein, a polymerase, dNTPs, ATP, a primer, and a template nucleicacid. In some embodiments, a RPA reaction can include one or more of thefollowing (in any combination): at least one recombinase; at least onesingle-stranded DNA binding protein; at least one DNA polymerase; dNTPsor a mixture of dNTPs and ddNTPs; a crowding agent; a buffer; a reducingagent; ATP or ATP analog; at least one recombinase loading protein; afirst primer and optionally a second primer; a probe; a reversetranscriptase; and a template nucleic acid molecule, e.g., asingle-stranded (e.g., RNA) or double stranded nucleic acid. In someembodiments, the RPA reactions can contain, e.g., a reversetranscriptase. Additional non-limiting examples of RPA reaction mixturesare described herein.

In some embodiments, the RPA reactions can contain a UvsX protein, agp32 protein, and a UvsY protein. Any of the processes, compositions orparticles described herein can contain, in part, e.g., a UvsX protein, agp32 protein, and a UvsY protein. For example, any of the processes,compositions, or particles described herein can contain, in part, T6H66SUvsX, Rb69 gp32, and Rb69 UvsY.

In some embodiments, the RPA reactions can contain a UvsX protein and agp32 protein. For example, any of the processes, compositions, orparticles described herein can contain, in part, e.g., a UvsX proteinand a gp32 protein.

One protein component of an RPA reaction is a recombinase, which mayoriginate from prokaryotic, viral or eukaryotic origin. Exemplaryrecombinases include RecA and UvsX (e.g., a RecA protein or UvsX proteinobtained from any species), and fragments or mutants thereof, andcombinations thereof. The RecA and UvsX proteins can be obtained fromany species. RecA and UvsX fragments or mutant proteins can also beproduced using the available RecA and UvsS protein and nucleic acidssequences, and molecular biology techniques (see, e.g., the mutant formsof UvsX described in U.S. Pat. No. 8,071,308). Exemplary UvsX proteinsinclude those derived from myoviridae phages, such as T4, T2, T6, Rb69,Aeh1, KVP40, Acinetobacter phage 133, Aeromonas phage 65, cyanophageP-SSM2, cyanophage PSSM4, cyanophage S-PM2, Rb14, Rb32, Aeromonas phage25, Vibrio phage nt-1, phi-1, Rb16, Rb43, Phage 31, phage 44RR2.8t,Rb49, phage Rb3, and phage LZ2. Additional exemplary recombinaseproteins include archaebacterial RADA and RADB proteins and eukaryotic(e.g., plant, mammal, and fungal) Rad51 proteins (e.g., RAD51, RAD51B,RAD51C, RAD51D, DMC1, XRCC2, XRCC3, and recA) (see, e.g., Lin et al.,Proc. Natl. Acad. Sci. U.S.A. 103:10328-10333, 2006).

In any process of this disclosure, the recombinase (e.g., UvsX) may be amutant or hybrid recombinase. In some embodiments, the mutant UvsX is anRb69 UvsX that includes at least one mutation in the Rb69 UvsX aminoacid sequence, wherein the mutation is selected from the groupconsisting of (a) an amino acid which is not histidine at position 64, aserine at position 64, the addition of one or more glutamic acidresidues at the C-terminus, the addition of one or more aspartic acidresidues at the C-terminus, and a combination thereof. In otherembodiments, the mutant UvsX is a T6 UvsX having at least one mutationin the T6 UvsX amino acid sequence, wherein the mutation is selectedfrom the group consisting of (a) an amino acid which is not histidine atposition 66; (b) a serine at position 66; (c) the addition of one ormore glutamic acid residues at the C-terminus; (d) the addition of oneor more aspartic acid residues at the C-terminus; and (e) a combinationthereof. Where a hybrid recombinase protein is used, the hybrid proteinmay, for example, be a UvsX protein that includes at least one regionthat includes an amino acid sequence derived from a different UvsXspecies. The region may be, for example, the DNA-binding loop-2 regionof UvsX.

Additionally, one or more single-stranded DNA binding proteins can beused to stabilize nucleic acids during the various exchange reactionsthat are ongoing in the reaction. The one or more single-stranded DNAbinding proteins can be derived or obtained from any species, e.g., froma prokaryotic, viral or eukaryotic species. Non-limiting exemplarysingle-stranded DNA binding proteins include E. coli SSB and thosederived from myoviridae phages, such as T4, T2, T6, Rb69, Aeh1, KVP40,Acinetobacter phage 133, Aeromonas phage 65, cyanophage P-SSM2,cyanophage PSSM4, cyanophage S-PM2, Rb14, Rb32, Aeromonas phage 25,Vibrio phage nt-1, phi-1, Rb16, Rb43, Phage 31, phage 44RR2.8t, Rb49,phage Rb3, and phage LZ2. Additional examples of single-stranded DNAbinding proteins include A. denitrificans Alide_2047, Burkholderiathailandensis BthaB_33951, Prevotella pallens HMPREF9144_0124, andeukaryotic single-stranded DNA binding protein replication protein A.

The DNA polymerase may be a eukaryotic or prokaryotic polymerase.Examples of eukaryotic polymerases include pol-alpha, pol-beta,pol-delta, pol-epsilon, and mutants or fragments thereof, orcombinations thereof. Examples of prokaryotic polymerase include E. coliDNA polymerase I (e.g., Klenow fragment), bacteriophage T4 gp43 DNApolymerase, Bacillus stearothermophilus polymerase I large fragment,Phi-29 DNA polymerase, T7 DNA polymerase, Bacillus subtilis Pol I,Staphylococcus aureus Pol I, E. coli DNA polymerase I, E. coli DNApolymerase II, E. coli DNA polymerase III, E. coli DNA polymerase IV, E.coli DNA polymerase V, and mutants or fragments thereof, or combinationsthereof. In some embodiments, the DNA polymerase lacks 3′-5′ exonucleaseactivity. In some embodiments, the DNA polymerase has strand-displacingproperties, e.g., large fragments of prokaryotic polymerases of class Ior pol V.

Any of the process of this disclosure may be performed in the presenceof a crowding agent. In some embodiments, the crowding agent may includeone or more of polyethylene glycol, polyethylene oxide, polyvinylalcohol, polystyrene, Ficoll, dextran, poly(vinylpyrrolidone) (PVP), andalbumin. In some embodiments, the crowding agent has a molecular weightof less than 200,000 daltons. Further, the crowding agent may bepresent, e.g., in an amount of about 0.5% to about 15% weight to volume(w/v).

If a recombinase loading protein is used, the recombinase loadingprotein may be of prokaryotic, viral or eukaryotic origin. Exemplaryrecombinase loading proteins include E. coli RecO, E. coli RecR, UvsY,and mutants or fragments thereof, or combinations thereof. ExemplaryUvsY proteins include those derived from myoviridae phages, such as T4,T2, T6, Rb69, Aeh1, KVP40, Acinetobacter phage 133, Aeromonas phage 65,cyanophage P-SSM2, cyanophage PSSM4, cyanophage S-PM2, Rb14, Rb32,Aeromonas phage 25, Vibrio phage nt-1, phi-1, Rb16, Rb43, Phage 31,phage 44RR2.8t, Rb49, phage Rb3, and phage LZ2. In any of the processesof this disclosure, the recombinase loading agent may be derived from amyoviridae phage. The myoviridae phage may be, for example, T4, T2, T6,Rb69, Aeh1, KVP40, Acinetobacter phage 133, Aeromonas phage 65,cyanophage P-SSM2, cyanophage PSSM4, cyanophage S-PM2, Rb14, Rb32,Aeromonas phage 25, Vibrio phage nt-1, phi-1, Rb16, Rb43, Phage 31,phage 44RR2.8t, Rb49, phage Rb3, or phage LZ2.

Further, any of the processes of this disclosure may be performed with ablocked primer. A blocked primer is a primer which does not allowelongation with a polymerase. Where a blocked primer is used, anunblocking agent can be used to unblock the primer to allow elongation.The unblocking agent may be an endonuclease or exonuclease which cancleave the blocking group from the primer. Exemplary unblocking agentsinclude E. coli exonuclease III and E. coli endonuclease IV.

In some embodiments, the processes of this disclosure can include:contacting a recombinase with a first and a second nucleic acid primerand a third extension blocked primer which contains one or morenoncomplementary or modified internal residue to form a first, second,and third nucleoprotein primer; contacting the first and secondnucleoprotein primers to the double stranded target nucleic acid to forma first double stranded structure between the first nucleoprotein primerand the first strand of DNA at a first portion of the first strand(forming a D loop) and a second double stranded structure between thesecond nucleoprotein primer and the second strand of DNA at a secondportion of the second strand (forming a D loop), such that the 3′ endsof the first nucleoprotein primer and the second nucleoprotein primerare oriented toward each other on the same target nucleic acid moleculewith a third portion of target nucleic acid present between the 5′ endsof the first and second primer; and extending the 3′ end of the firstnucleoprotein primer and second nucleoprotein primer with one or morepolymerases and dNTPs to generate a first amplified target nucleic acid;contacting the first amplified target nucleic acid to the thirdnucleoprotein primer to form a third double stranded structure in thefirst amplified target nucleic acid (forming a D loop) in the presenceof a nuclease, wherein the nuclease specifically cleaves thenoncomplementary internal residue only after the formation of the thirddouble-stranded structure to form a third 5′ primer and a third 3′extension blocked primer; and extending the 3′ end of the third 5′primer with one or more polymerase and dNTP to generate a seconddouble-stranded amplified nucleic acid.

In some embodiments, the processes include a first and second primer toamplify a first portion present within a double-stranded target nucleicacid to generate a first amplified product, and at least one additionalprimer that can be used to amplify a contiguous sequence present withinthe first amplified product (e.g., an additional third primer that canbe used in combination with, e.g., the first or the second primer, toamplify a contiguous sequence present within the first amplifiedproduct). In some embodiments, the processes include a first and secondprimer to amplify a first portion present within a double-strandedtarget nucleic acid to generate a first amplified product, and a thirdand fourth primer that can be used to amplify a contiguous sequencepresent within the first amplified product.

In some embodiments, the processes can include, e.g., a forward primerand a reverse primer. In some embodiments, the processes can include atleast one blocked primer which comprises one or more noncomplementary ormodified internal residues (e.g., one or more noncomplementary ormodified internal residues that can be recognized and cleaved by anuclease, e.g., DNA glycosylase, AP endonuclease, fpg, Nth, MutY, MutS,MutM, E. coli. MUG, human MUG, human Ogg1, a vertebrate Nei-like (Neil)glycosylase, Nfo, exonuclease III, or uracil glycosylase). Additionalnon-limiting examples of nucleic acids (e.g., primers and probes) thatcan be included in a process are described herein.

In some embodiments, the processes can include a primer or probe that isnuclease resistant, e.g., a primer or probe that contains at least one(e.g., at least two, three, four, five, six, seven, or eight)phosphorothioate linkages.

Any of the processes of this disclosure may be performed in the presenceof heparin. Heparin may serve as an agent to reduce the level ofnon-specific primer noise, and to increase the ability of E. coliexonuclease III or E. coli endonuclease IV to rapidly polish 3′ blockinggroups or terminal residues from recombination intermediates.

Based on the particular type of reaction, the mixture can also containone or more of buffers, salts, and nucleotides. The reaction mixture canbe maintained at a specific temperature or temperature range appropriateto the reaction. In some embodiments, the temperature is maintained ator below 80° C., e.g., at or below 70° C., at or below 60° C., at orbelow 50° C., at or below 40° C., at or below 37° C., at or below 30°C., or at or below room temperature. In some embodiments, thetemperature is maintained at or above 4° C., at or above 10° C., at orabove 15° C., at or above 20° C., at or above room temperature, at orabove 25° C., at or above 30° C., at or above 37° C., at or above 40°C., at or above 50° C., at or above 60° C., or at or above 70° C. Insome embodiments, the reaction mixture is maintained at room or ambienttemperature. In some embodiments, the Celsius-scale temperature of themixture is varied by less than 25% (e.g., less than 20%, less than 15%,less than 10%, or less than 5%) throughout the reaction time and/or thetemperature of the mixture is varied by less than 15° C. (e.g., lessthan 10° C., less than 5° C., less than 2° C., or less than 1° C.)throughout the reaction time.

Detection of amplification, e.g., in real time, may be performed by anymethod known in the art. In some embodiments, one or more primers orprobes (e.g., molecular beacon probes) are labeled with one or moredetectable labels. Exemplary detectable labels include enzymes, enzymesubstrates, coenzymes, enzyme inhibitors, fluorescent markers,quenchers, chromophores, magnetic particles or beads, redox sensitivemoieties (e.g., electrochemically active moieties), luminescent markers,radioisotopes (including radionucleotides), and members of bindingpairs. More specific examples include fluorescein, phycobiliprotein,tetraethyl rhodamine, and beta-galactosidase. Binding pairs may includebiotin/avidin, biotin/strepavidin, antigen/antibody, ligand/receptor,and analogs and mutants of the binding pairs.

It should be noted that a fluorescence quencher is also considered adetectable label. For example, the fluorescence quencher may becontacted to a fluorescent dye and the amount of quenching is detected.

Particle Detection

Detection and monitoring of the particles can be performed using anysuitable method. Exemplary methods include microscopy, light scattering,flow cytometry, and microfluidic methods.

In some embodiments, the particles can be detected using microscopy,e.g., differential interference contrast or fluorescence microscopy, todirectly observe the particles at high magnification. With the aid of acomputer, microscope images can be automatically obtained and analyzed.Additionally, microscopy can allow for continual or frequent monitoringof at least a portion of a mixture containing particles.

In some embodiments, the particles can be detected using flow cytometry.In flow cytometry, one or more beams of light, e.g., each of a singlewavelength, are directed onto a hydrodynamically-focused stream offluid. Suspended particles passing through the beams scatter the light,and fluorescent chemicals found in the particle or attached to theparticle may be excited. The scattered and/or fluorescent light ispicked up by detectors within the device, from which information aboutparticle size and fluorescence can be determined. Modern flow cytometerscan analyze several thousand particles every second, in “real time,” andcan actively separate and isolate particles having specified properties.

In some embodiments, the particles can be detected using cytometrymethods, devices, and systems as disclosed in US 2009/0079963, US2010/0179068, and WO 2009/112594.

In some embodiments, the particles can be detected using microfluidicmethods, devices, and systems. For example, the particles can bedetected using a lab-on-a-chip device or system, or the like. See, e.g.,U.S. Patent Application Publication Nos. 2009/0326903 and 2009/0297733

In some embodiments, the particles can be detected using a device orsystem suitable for point-of-care, field, or consumer use. For example,a device (e.g., a lap-on-a-chip device) can include a recombinase, apolymerase, a single-stranded binding protein, ATP, dNTPs, and a primeror probe. In some embodiments, a device can be provided that contains arecombinase, a polymerase, a single-stranded binding protein, ATP,dNTPs, and a primer or probe, where one of the recombinase, thepolymerase, the primer or probe, or recombinase is covalently attachedor non-covalently bound (e.g., through use of an affinity tag) to asurface. In some embodiments, particles can be placed in multiple singlewells in a multi-well plate.

In any of the disclosed methods, where desired the particles may befixed prior to detection. For example, the particles can be fixed bytreatment with an aldehyde (e.g., formaldehyde, paraformaldehyde, orglutaraldehyde) to cross-link proteins and nucleic acids in the sample,effectively stopping the progress of reactions in the mixture andallowing for observation of the particles in the state at which thereaction was stopped. By fixing the mixtures, the particles may bedetected at a later point in time, potentially simplifying processingand detection.

Oligonucleotides

Oligonucleotides as disclosed herein may serve as amplification primersand/or detection probes. In some embodiments, the oligonucleotides areprovided as a set of two or more (e.g., two, three, four, or more)oligonucleotides, e.g., for use in an amplification method (e.g., asdescribed herein).

Oligonucleotides can be synthesized according to standardphosphoroamidate chemistry, or otherwise. Modified bases and/or linkerbackbone chemistries may be desirable and functional in some cases andcan be incorporated during synthesis. Additionally oligonucleotides maybe modified with groups that serve various purposes e.g. fluorescentgroups, quenchers, protecting (blocking) groups (reversible or not),magnetic tags, proteins etc.

In some embodiments, the oligonucleotide used herein can contain acontiguous sequence (e.g., at least 10 base units) that is at least 90%identical (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) to a contiguous sequence present within a target nucleic acid.The percent identity or homology between two sequences can be determinedusing a mathematical algorithm. A non-limiting example of a mathematicalalgorithm utilized for the comparison of two sequences is the algorithmof Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA, 87:2264-68,modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA,90:5873-77. Such an algorithm is incorporated into the NBLAST program ofAltschul, et al., (1990); J. Mol. Biol. 215:403-410. To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al., (1997) Nucleic Acids Res. 25:3389-3402.When utilizing BLAST and Gapped BLAST programs, the default parametersof the NBLAST program can be used. See online at ncbi.nlm.nih.gov.

The oligonucleotides may include one or more detectable labels. Thedetectable label may be a fluorophore, an enzyme, a quencher, an enzymeinhibitor, a radioactive label, a redox sensitive moiety (e.g., anelectrochemically active moiety) one member of a binding pair and acombination thereof. In some embodiments, the oligonucleotides caninclude both a fluorophore and a quencher. The quencher may be close tothe fluorophore to suppress the fluorescence of the fluorophore. Forexample, the separation between the fluorophore and the quencher may be0 to 2 bases, 0 to 5 bases, 0 to 8 bases, 0 to 10 bases, 3 to 5 bases, 6to 8 bases, and 8 to 10 bases. The fluorophore and quencher may be anyfluorophore and quencher known to work together including, but notlimited to, the fluorophore and quenchers any of the fluorophoresdescribed in this disclosure. Where the detectable label is afluorophore or a quencher, it may be attached to the oligonucleotide bya fluorophore-dT amidite residue or quencher-dT amidite residuerespectively. Other attachments are possible and widely known.

In another aspect, either the fluorophore or the quencher may beattached to a modified internal residue and the fluorophore and quenchercan be separated following cleavage of the modified internal residue bythe nuclease.

While any fluorophore may function for the methods of the invention,fluorescein, FAM, TAMRA, and Texas Red are exemplary fluorophores.Exemplary quenchers include a dark quencher which may be, for example,Dark Quencher 1, Dark Quencher 2, Black Hole Quencher 1 or Black HoleQuencher 2.

In some embodiments, the oligonucleotides can include a modifiedinternal residue. The modified internal residue may be any chemicalstructure (residue) that cannot form a Watson-Crick base pairingstructure with its corresponding base in a double stranded nucleic acidstructure. The term “modified internal residue,” also includes, atleast, any residue not normally found in DNA—that is any residue whichis not an “A”, “G”, “C” or “T” such as, for example uracil or inosine.In some embodiments, the modified internal residue is inosine, uracil,8-oxoguanine, thymine glycol, or an abasic site mimic. Preferred abasicsite mimics include a tetrahydrofuran residue or D-spacer (which can beproduced as a product of employing a5′-O-Dimethoxytrityl-1′,2′-Dideoxyribose-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramiditeduring oligonucleotide synthesis.

In some embodiments, the oligonucleotides are extension blocked. Anextension blocked oligonucleotide is blocked at its 3′ end so that itcannot normally be elongated by polymerase and dNTP even in the presenceof a complimentary template. Methods of blocking an oligonucleotide arewell known and include, at least, the inclusion of a blocked 3′nucleotide. The blocked 3′ nucleotide may contain, for example, ablocking group that prevents polymerase extension. Generally, theblocking groups are attached to the 3′ or 2′ site of the 3′ sugarresidue but other locations of attachments are possible. One of the mostcommon 3′ blocking methods is to place a dideoxy sugar at the 3′ end ofan oligonucleotide. The blocking group may be, for example, a detectablelabel.

In some embodiments, the oligonucleotides disclosed herein may bemodified by incorporation of one or more detectable labels, modifiedresidues (e.g., modified internal residues), and blocking groups. Whenthe oligonucleotide disclosed herein includes one or more detectablelabels, modified residues (e.g., modified internal residues), andblocking groups, the oligonucleotide without such modifications or withadditional modifications is also included in the disclosure.Additionally, an oligonucleotide as disclosed herein that includes oneor more detectable labels, modified residues (e.g., modified internalresidues), and blocking groups may have such a moiety replaced byanother detectable label, modified residue (e.g., modified internalresidue), or blocking group, e.g., a detectable label, modified residue(e.g., modified internal residue), or blocking group as disclosedherein.

Applications

The methods and compositions disclosed herein can be used, for example,to detect the number of copies of a target nucleic acid and to monitoramplification of a sequence present within a target nucleic acid. Insome embodiments of the present methods, the target nucleic acids can bedetected at low copy numbers and in relatively crude samples. In someembodiments, the detected nucleic acid is a bacterial nucleic acid,e.g., from a bacterium selected from Chlamydia trachomatis, Neisseriagonorrhea, Group A Streptococcus, Group B Streptococcus, Clostridiumdifficile, Escherichia coli, Mycobacterium tuberculosis, Helicobacterpylori, Gardnerella vaginalis, Mycoplasma hominis, Mobiluncus spp.,Prevotella spp., and Porphyromonas spp, or from another bacteriumdescribed herein or known in the art. In some embodiments, the detectednucleic acid is a mammalian nucleic acid, e.g., a nucleic acid isassociated with tumor cells. In some embodiments, the detected nucleicacid is a viral nucleic acid, e.g., from HIV, influenza virus, or denguevirus, or from another virus. In some embodiments, the detected nucleicacid is a fungal nucleic acid, e.g., from Candida albicans or anotherfungus. In some embodiments, the detected nucleic acid is a protozoannucleic acid, e.g., from Trichomonas or another protozoan. The methodsand compositions disclosed herein can be used in the diagnosis of adisorder or state associated with a detected nucleic acid, e.g., abacterial nucleic acid, mammalian nucleic acid, viral nucleic acid,fungal nucleic acid, or protozoan nucleic acid (e.g., as disclosedherein). For example, the methods and compositions provided herein canbe used to diagnose a bacterial infection, a viral infection, a fungalinfection, or a parasitic infection. In some embodiments, the detectednucleic acid is a nucleic acid from: influenza A or a variant thereof,influenza B or a variant thereof, methicillin-resistant Staphylococcusaureus (MRSA), C. difficile, M tuberculosis, Chlamydia species (e.g.,Chlamydia trachomatis), N. gonorrhoeae, Treponema pallidum, humanpapilloma virus (HPV) (e.g., HPV variants type 16 and type 18),hepatitis virus (e.g., hepatitis A, B, and C), or a circulating cancercell. In some embodiments, the methods and compositions provided hereincan be used to diagnose MRSA infection, C. difficile infection,tuberculosis, chlamydia infection, gonorrhea, syphilis, HPV infection,hepatitis viral infection, or HPV infection. The methods andcompositions disclosed herein can be used in quantification of nucleicacids. “Digitalization” of nucleic acid amplification/detectionreactions is a recent approach to allow for accurate counting oftemplate molecules (see, e.g., Vogelstein, 1999, Proc. Natl. Acad. Sci.USA, 96:9236). Typically in these methods, spatial separation of thereaction mixture into the required micro-compartments (typically in thenanoliter range) is achieved by physically splitting an amplificationreaction, e.g. by pressing it under pressure into suitable microfluidiccassettes or by dispersing it in a suitable emulsion. Without wishing tobe bound by theory, if the particles disclosed herein are active centersof amplification, then the presence of the particles constitutes aninherent compartmentalization of the reaction mixture that may be usedin quantification. For example, by counting the number of “active” RPAparticles (e.g., those associated with the generation of a fluorescentsignal) one can measure or estimate the number of template nucleic acidmolecules present in the reaction mixture.

The methods can also be used to detect the physical linkage of two ormore nucleic acids. In many molecular biology applications the detectionof physical linkage of two different genetic markers present in a givensample is important. For example, the mere presence of a bacterialspecies marker and an antibiotic-resistance marker in a given sampledoes not deliver information about whether both markers are present inthe same bacteria (e.g., on the same nucleic acid), or whether themarkers are present in separate co-colonizing bacteria species.Demonstrating that the two markers are linked on a single piece ofgenomic DNA associates the antibiotic resistance with a particularpathogen. The co-localization of the two markers can deliver vitaldiagnostic information in this scenario.

The methods and compositions described herein can be used to demonstratethat the sites or locations of two amplification events for two nucleicacids are overlapping, providing information about the physical linkageof the nucleic acids. In contrast, separable amplification events canindicate the presence of both nucleic acids, but on separate segments ofDNA (e.g., in two co-colonizing species of bacteria). In someembodiments, the linkage of two nucleic acid sequences can be detectedby observing active amplification products of both localized to a singleparticle in a reaction mixture. In other embodiments, the linkage of twonucleic acid sequences on a single segment of DNA can be detected byobserving “tethering” of two particles, each amplifying one of thenucleic acids, by the DNA segment.

In some embodiments, observation of the particles disclosed herein canbe used in methods of quality control. For example, a relationshipbetween particle appearance (number, size, density) and RPA performancecan be used to generate an analytical parameter to predict RPA reactionquality prior to amplification. This could be used for general qualitycontrol purposes (e.g., to check what type/number of particles arepresent in a given reaction mixture), or to monitor the effect ofchanges in production procedures (e.g., stabilization processes) or instorage conditions, etc.

In some embodiments, the methods and compositions disclosed herein canbe used to obtain results of amplification reactions within minutes(e.g., within 8, 7, 6, 5, 4, 3, 2, 1.5, or 1 minute) from the start ofthe reaction. Typically, monitoring amplification reactions by detectingthe accumulation of fluorescence signal is performed “in bulk”, i.e. thesignals generated by individual template molecules is integrated overthe entire given reaction volume, producing a detectable fluorescenceresponse in 5-8 minutes. In contrast, observing the fluorescence signalgenerated at RPA particles may also in principle be used to shorten thetime to result in a reaction. This result is due, at least in part, tohigher sensitivity of detection under high magnification in defined loci(e.g., particles).

The fluorescence signal strength of standard RPA reactions, typicallyperformed and monitored ‘in bulk’, does profit from mixing stepsperformed during the incubation, especially if very low amounts ofstarting template material are used. Observing amplification reactionsdirectly at particles can reduce any variation introduced by mixing.

EXAMPLES Example 1 Particles in Recombinase Polymerase AmplificationMixtures

This example describes the observation of particles containingoligonucleotides within RPA mixtures. Freeze dried mixtures of RPAreaction components including FAM labeled oligonucleotides were obtainedby preparing a mixture containing 2.96 μg Creatine Kinase, 13.1 μg Rb69gp32, 18.1 μg T6 H66S UvsX, 5.15 μg Rb69 UvsY, 5.38 μg Exonuclease IIIand 5.0 μg DNA Polymerase (large fragment of S. aureus polymerase I) in80 μl 9.38 mM Tris Acetate, pH 8.3, 3.13 mM DTT, 2.5% PEG, 3.75%trehalose, 31.3 mM phosphocreatine, 1.56 mM ATP, 750 μM dNTPmix (188 μMeach of dATP, dTTP, dCTP and dGTP), 388 nM Spy1258F2 (CACAGACACTCGACAAGTCCTCAATCAAACCTTG; SEQ ID NO:1), 363 nM Spy1258R2 (CAGAAATCCTTGATGAGTTGCGGAAATTTGAGGT; SEQ ID NO:2) and 75 nM Spy1258exoP1(CCTTGTCCTACCTTATAGAACATAGAGAATQTHFAACCGCACTCGCTAC; F=FAM-dT, H=THF(abasic site mimic), Q=BHQ-1-dT, 3′=block c3spacer; SEQ ID NO:3) andfreeze-drying the mixture in 0.2-mL tubes. The dried reagents wereresuspended in 46.5 μL rehydration buffer (48 mM Tris acetate, 133.8 mMKOAc, 2% PEG)+3.5 μL water and vortexed. These mixtures did not containnucleic acid template or magnesium. Ten microliters of the mixture wastransferred to a microscope slide and imaged using differentialinterference contrast (DIC) and fluorescence microscopy at 40×magnification (FIG. 1 (A-C)). Particles of about 1-10 microns in sizewere observed using DIC (FIG. 1 (A)) or fluorescence (FIG. 1 (B)), andwhen the two images were merged (FIG. 1 (C)). Approximately 100-500particles/nL were observed (field of view at 40× magnification wasequivalent to 1.55 nL of the mixture).

In a separate experiment, mixtures were prepared as above butsubstituting 2.5 μL water and 1 μL of a Streptococcus pyogenes genomicDNA preparation (100 copies/μl) for the 3.5 μL water. The mixture wasvortexed and imaged as above (FIG. 2 (A-C)). Similar particles as abovewere observed using DIC (FIG. 2 (A)) or fluorescence (FIG. 2 (B)), andwhen the two images were merged (FIG. 2 (C)).

This example demonstrates that particles are formed in RPA mixtures, andthat the particles are not dependent upon the inclusion of template ormagnesium.

Example 2 Crowding Agents Stimulate Particle Formation

To determine the effects of crowding agents on particle formation, freshRPA mixtures were prepared containing 2.96 μg Creatine Kinase, 13.1 μgRb69 gp32, 18.8 μg T6 H66S UvsX, 2.5 μg Rb69 UvsY, 5.38 μg ExonucleaseIII and 5.0 μg DNA Polymerase in 50 mM Tris Acetate, pH 8.3, 100 mMKOAc, 5 mM DTT, 1.2 mM dNTP mix (300 μM each of dATP, dTTP, dCTP anddGTP), 50 mM phosphocreatine, 2.5 mM ATP, 6% trehalose, 14 mM MgAc, 30nM HIV p2LFtexas (Texas red-labeled oligoAGAATTACAAAAACAAATTACAAAAATTCA5AATTTTCGGGTTT; 3′ dA blocked, 5′ TexasRed, 5=dSpacer; SEQ ID NO:4), 420 nM Spy1258F2(CACAGACACTCGACAAGTCCTCAATCAAACCTTG; SEQ ID NO:5), and 390 nM Spy1258R2(CAGAAATCCTTGATGAGTTGCGGAAATTTGAGGT; SEQ ID NO:6). PEG was included ineach mixture at 0%, 2%, 2.5%, 3%, 3.5%, 4%, 5.5%, or 8%. The mixtureswere mixed by pipette and 10 μL of each was transferred to a microscopeslide. Imaging was performed using differential interference contrast(DIC) and fluorescence microscopy at 40× magnification. The number ofparticles observed increased with increasing PEG concentration up to5.5% (FIG. 3 (A-G)). Fewer particles were observed at 8% PEG (FIG. 3(H)).

This example demonstrates that PEG can enhance formation of particles inRPA mixtures.

Example 3 Contribution of Mixture Components to Particle Formation

To determine the contribution of the RPA mixture components to particleformation, mixtures were prepared as in Example 2 with 5.5% PEG, exceptthat individual components were excluded in each reaction. The mixtureswere imaged as above using DIC and fluorescence microscopy. When all thecomponents were present, particles formed in the mixture as describedabove (FIG. 4 (A-B)). Particles formed in the absence of UvsX appeareddifferent in size from those formed in the presence of UvsX and were noteasily observable by DIC (FIG. 4 (C-D)). Particles formed in the absenceof gp32 appeared different in shape and size from those formed in thepresence of gp32 (FIG. 4 (E-F)). Structures formed in the absence ofother RPA components (UvsY, DNA polymerase, creatine kinase, orexonuclease III) appeared similar to those formed in a complete RPAreaction (FIG. 5 (A-H)). The absence of UvsY did appear to lead to aslight decrease in the number of the particles and an increase in theparticle size (FIG. 5 (A-B)).

Additional mixtures were prepared excluding two or three reactioncomponents. A control RPA mixture was prepared containing 2.96 μgCreatine Kinase, 13.1 μg Rb69 gp32, 18.8 μg T6 H66S UvsX, 5.15 μg UvsY,8.26 μg Exonuclease III and 5.0 μg DNA Polymerase in 50 mM Tris Acetate,pH 8.3, 100 mM KOAc, 5 mM DTT, 1.2 mM dNTP mix (300 μM each of dATP,dTTP, dCTP and dGTP), 50 mM phosphocreatine, 2.5 mM ATP, 6% trehalose,14 mM MgAc, 5.5% PEG, 120 nM M2intFAM (FAM-labeled probe5′-tcctcatatccattctgTcgaatatcatcaaaagc-3′; T=carboxyfluorescein-dT; SEQID NO:19), 420 nM each SpaF3 (CGCTTTGTTGATCTTTGTTGAAGTTATTTTGTTGC; SEQID NO:7) and SpaR10+1 (TTAAAGATGATCCAAGCCAAAGTCCTAACGTTTTA; SEQ IDNO:8). Parallel mixtures were prepared that lacked (i) gp32 and UvsY;(ii) UvsX and UvsY; (iii) UvsX and gp32; (iv) UvsX, UvsY, and gp32; or(iv) gp32, UvsY, and Emix (phosphocreatine and ATP). No particles wereobserved in the mixtures lacking UvsX and at least one other component.In the mixtures lacking gp32 and UvsY, large, irregular fluorescentbodies were observed (FIG. 6 (A-C)).

This example demonstrates that exclusion of UvsX or gp32 has the largesteffect on particle morphology, followed by an intermediate effect ofexclusion of UvsY, with no significant effect observed on exclusion ofDNA polymerase, creatine kinase, or exonuclease III. Exclusion of two ormore components had increased effects.

Example 4 Separate Populations of Particles Remain Distinct when Mixed

Two freeze-dried mixtures were prepared as described in Example 1,except that each mixture included different oligonucleotides and 18.8 μgUvsX. Reagent Mix 1 contained 296 nM SpaF3 (SEQ ID NO:7), 298 nMSpaR10+1 (SEQ ID NO:8) and 149 nM SpaProbe1(CATCAGCTTTTGGAGCTTGAGAGTCAT9 A8G6TTTTGAGCTTCAC; 3′ biotin, 6=BHQ-2 dT,8=dSpacer, 9=TMR dT; SEQ ID NO:9). Reagent Mix 2 contained 299 nMMecF9-8+2 (CCCTCAAACAGGTGAA TTATTAGCACTTGT; SEQ ID NO:10), 300 nM MecR1a(CTTGTTGAGCAGAGG TTCTTTTTTATCTTC; SEQ ID NO:11) and 150 nM MecProbe1(ATGACGTCTAT CCATTTATGTATGGCAFGHGQAACGAAGAATATA; 3′ biotinTEG, Q=BHQ-1dT, H=THF (abasic site mimic), F=FAM-dT; SEQ ID NO:12). Equal volumes ofthe two reagent mixtures were combined, and 80 μL was dispensed into0.2-mL tubes and freeze-dried. The dried mixtures were resuspended in46.5 μl rehydration buffer (see Example 1), 1 μL water, and 2.5 μL 280mM MgAc. The mixture was vortexed and 10 μL was transferred to amicroscope slide for imaging using DIC and fluorescence. The particlesobserved contained both red (TMR) and green (FAM) fluorescence,indicating that both labeled oligonucleotides were present in theparticles (FIG. 7 (A-F)).

In another experiment, two separate freeze-dried mixtures were preparedas above, one including only the TMR labeled Spa RPA probe (Reagent Mix1, above), and the other including only the FAM-labeled MecA RPA probe(Reagent Mix 2, above). Following reconstitution, the two reconstitutedmixtures were combined and imaged using DIC and fluorescence. Distinctparticles that contained predominantly one fluorophore or the other wereobserved in the mixture (FIG. 8 (A-F)). This indicates that theparticles including each probe can remain distinct from each other aftermixing.

To determine the stability of mixed populations of particles over time,two primer-free freeze-dried reactions were reconstituted in rehydrationbuffer with MgAc and oligonucleotides as below. One mixture included 30nM HIV p2LFtexas (Texas Red labeled), 420 nM Spy1258F2 (SEQ ID NO:1,unlabeled), and 390 nM Spy1258R2 (SEQ ID NO:2, unlabeled). The othermixture included 50 nM M2intFAM oligo (SEQ ID NO:19, FAM labeled), 420nM Spy1258F2 (SEQ ID NO:1, unlabeled), and 390 nM Spy1258R2 (SEQ IDNO:2, unlabeled). Five microliters of each mixture were pipetted onto amicroscope slide and mixed, and the combination was imaged at 2, 7, and13 minutes (FIG. 9). Images of the mixture following the 12-minuteperiod are shown in FIG. 9. After 13 minutes, particles includingpredominantly Texas Red or FAM fluorescence were observable.

This example demonstrates that particles remain relatively stable insolution and can be independently labeled. This observation can beuseful in monitoring two or more RPA reactions simultaneously, occurringon different particle subsets.

Example 5 RPA Reactions are Observed Localized to Particles

Freeze dried mixtures of RPA reaction components including a FAM labeledoligonucleotide probe, as in Example 1, were reconstituted with 46.5 μlrehydration buffer, and an amplification reaction was begun by additionof 1 μL 50,000 copies/4 S. pyogenes genomic DNA and 2.5 μL 280 mM MgAc.The reaction was mixed by pipetting and transferred to a microscopeslide for imaging by DIC and fluorescence starting at about 2 minutes,40 seconds after initiation and then at 8, 12, 14, 15, 16, 18, 20, and22 minutes (FIG. 10). An increase in fluorescence (indicatingamplification) was observed, which was at least initially localized toindividual particles.

In another experiment, freeze-dried primer-free mixtures of reactioncomponents (prepared by mixing a 50 μl volume of 2.96 μg CreatineKinase, 9.88 μg Rb69 gp32, 18.8 μg T6 H66S UvsX, 5.38 μg UvsY, 5.38 μgExonuclease III and 5.34 μg DNA Polymerase in 25 mM Tris Acetate, pH8.3, 5 mM DTT, 2.28% PEG, 5.7% trehalose, 50 mM phosphocreatine, 2.5 mMATP, 1200 μM dNTPmix (300 μM each of dATP, dTTP, dCTP and dGTP andfreeze drying in 0.2-mL tubes) were reconstituted with 29.5 μlprimer-free rehydration buffer (41.7 mM Tris Acetate, 167.5 mM PotassiumAcetate, 5.4% PEG, pH 8.3), 3.5 μL of 6 μM Spa F3 (SEQ ID NO:7), 3.5 μLof 6 μM Spa R10+1 (SEQ ID NO:8), 1 μL of 6 μM TMR-labeled Spa Probe 1(SEQ ID NO:9), 1 μL of 0.6 μM M2intFAM oligo (SEQ ID NO:19, used as afluorescent marker of particles and not involved in the RPA reaction),and 8 μL water. The reaction was initiated by addition of 1 μL 50,000copies/4 Group A Streptococcus purified genomic template DNA and 2.5 μL280 mM MgAc and mixing by pipette. Ten microliters of the mixture weretransferred to a microscope slide, and imaging was begun about 3 minutesafter initiation of the reaction. A time course of the reaction mixtureat 3, 8, 15, 18, 22, and 26 minutes (FIG. 11) showed an increase in redfluorescence (indicating amplification), which was at least initiallylocalized to individual particles.

This example demonstrates that nucleic acid amplification products canbe observed colocalized with particles.

Example 6 Effects of UvsX Variants

To investigate the effects of different UvsX variants, mixtures were setup at room temperature containing 2.96 μg Creatine Kinase, 13.1 μg Rb69gp32, 8.26 μg Exonuclease III, 5.0 μg Polymerase in 50 mM Tris Acetate,pH 8.3, 100 mM KOAc, 5 mM DTT, 1.2 mM dNTP mix (300 μM each of dATP,dTTP, dCTP and dGTP), 50 mM phosphocreatine, 2.5 mM ATP, 6% trehalose,14 mM MgAc, 5.5% PEG, 120 nM M2intFAM (SEQ ID NO:19), 420 nM each SpaF3and SpaR10+1 (50 μl final volume). Four different mixtures wereprepared, containing either 18.8 μg T6H66S UvsX or 17.6 μg T6 UvsX, andwith or without 5.15 μg Rb69 UvsY. Ten microliters of each mixture weretransferred to a microscope slide and imaged at 20× magnification about5-20 minutes after set-up (FIG. 12 (A-D)) and also at 40× magnificationabout 50-60 minutes after set-up (FIG. 13 (A-D)). In general, moreparticles were observed in the T6H66S UvsX mixture than with T6 UvsX.Additionally the T6H66S UvsX particles were often different in shapethan those with T6 UvsX, including more comet-like shapes, whereas theT6 UvsX particles were more spherical. The T6H66S UvsX mixtures lackingUvsY had has more diffuse particles and diffuse “halos” or “doughnuts”that lacked signal in the middle. With T6 UvsX the opposite effect wasoften observed. Without UvsY, the particles were bright small spheres,but with UvsY they were less bright and more smeary and small.

The effect of UvsY on RPA reaction kinetics using T6H66S UvsX and T6UvsX was investigated. Reactions were prepared as above, with T6H66SUvsX or T6 UvsX, and with or without Rb69 UvsY. Three separateexperiments were performed using different primer sets and templates. Inthe first experiment, the primers were 420 nM FluAPAFNA507(AACCTGGGACCTTTGATCTTGGGGGGCTATATG; SEQ ID NO:13) and FluAPARNA106(ATGTGTTAGGAAGGAGTTGAACCAAGAAGCATT; SEQ ID NO:14), with 120 nM probeFluAPAexoP2.2 (GATCTTGGGGGGCTATATGAA GCAATYGAGGAGFHCQTGATTAATGAT;F=FAM-dT, H=THF (abasic site mimic), Q=BHQ-1-dT, 3′=block c3spacer; SEQID NO:15). Five hundred or 50 copies of influenza A template RNA wereused in each reaction. RPA reactions were assembled containing 2.96 μgCreatine Kinase, 13.1 μg Rb69 gp32, 18.8 μg T6H66S UvsX or 17.6 ug T6UvsX, 8.26 μg Exonuclease III, 5.0 μg Polymerase, 1.79 μg ReverseTranscriptase, 5.15 μg UvsY (when present) in 50 mM Tris Acetate, pH8.3, 100 mM KOAc, 5 mM DTT, 0.2 units/4 Ribolock™ RNAse Inhibitor(Fermentas), 1.2 mM dNTP mix (300 μM each of dATP, dTTP, dCTP and dGTP),50 mM phosphocreatine, 2.5 mM ATP, 6% trehalose, 5.5% PEG. The abovecomponents were assembled in a 46.5 μL volume in a 0.2-mL tube, andreactions were started by addition of 2.5 μL of 280 mM MgOAc and 1 μL of500 or 50 copies/4 influenza A template RNA. Reactions were vortexed,briefly centrifuged and transferred to a Twista instrument (ESE) and thefluorescence monitored every 20 seconds for 20 minutes at 40° C. with amixing step (vortex and brief centrifuge) at 5 minutes. Inclusion ofUvsY with T6H66S UvsX led to a delay in amplification relative to thereaction without UvsY, and the opposite was seen for T6 UvsX (FIG. 14(A-B)). When only 50 copies of the template were present, the reactionslacking UvsY generated less signal overall (FIG. 14 (A-B)). A similareffect on reaction kinetics was observed in a second experiment using asprimers 420 nM SpaF3 and SpaR10, with 120 nM SpaProbe1.2 and 1000 copiesof group A streptococcus purified genomic template DNA (FIG. 15).

In the second experiment, the primers used were 420 nM FluBNSF1007(CATCGGATCCTCAACTCACTCTTCGAGCGT; SEQ ID NO:16) and FluBNSR705(GACCAAATTGGGATAAGACTCCCACCGCAGTTTC; SEQ ID NO:17), with 120 nM probeFluBNSexoP1 (CATCGGATCCTCAAYTCACTCTTCGAGCGFHTQAA TGAAGGACATTC; F=FAM-dT,H=THF (abasic site mimic), Q=BHQ-1-dT, 3′=block c3spacer; SEQ ID NO:18).Five hundred copies of PCR-amplified influenza B template DNA were usedin each reaction. In these reactions, no amplification was observed withT6H66S UvsX lacking UvsY (FIG. 16 (A-B)). The opposite effect wasobserved with T6 UvsX (FIG. 16 (A-B)).

This example demonstrates that different UvsX variants can havedifferent requirements for UvsY with regard to particle morphology andamplification reaction kinetics. Additionally, particle morphologyappears to be correlated to the kinetics and/or progress of theamplification reaction.

Example 7 Quantification of Nucleic Acids

The methods disclosed herein can be used for the quantification ofnucleic acids. In one experiment, dilutions of a template nucleic acidare combined with an RPA reaction mixture as disclosed in Example 5. Thenumber of particles in a specified volume of the reaction that areassociated with sites of nucleic acid amplification is determined ateach dilution. Within a range, the number of particles associated withsites of nucleic acid amplification varies with the concentration oftemplate nucleic acid in the reaction. For example, the number ofparticles associated with nucleic acid amplification can be proportionalto the concentration of template nucleic acid, or the number ofparticles associated with nucleic acid amplification can be equivalentto the number of template nucleic acid molecules in the same volume.Using this information regarding the correlation between number ofactive particle and template nucleic acid concentration, theconcentration of template nucleic acid in an experimental sample can bedetermined.

OTHER EMBODIMENTS

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

What is claimed is:
 1. A process comprising: (a) providing a mixturecomprising a recombinase, a DNA polymerase, a template nucleic acid,dNTPs or a mixture of dNTPs and ddNTPs, a single-stranded DNA bindingprotein, and one or more oligonucleotides; and (b) detecting particlesassociated with nucleic acid amplification products in the reactionmixture by observing particles or determining the number or proportionof particles associated with the nucleic acid amplification products inthe reaction mixture.
 2. The process of claim 1, wherein the mixturecomprises a crowding agent.
 3. The process of claim 2, wherein thecrowding agent comprises polyethylene glycol, polyvinyl alcohol, dextranand/or Ficoll.
 4. The process of claim 2, wherein the crowding agentcomprises polyethylene glycol.
 5. The process of claim 3, wherein thepolyethylene glycol is selected from the group consisting of PEG1450,PEG3000, PEG8000, PEG10000, PEG14000, PEG15000, PEG20000, PEG250000,PEG30000, PEG35000, PEG40000, PEG compound with molecular weight between15,000 and 20,000 daltons, and combinations thereof.
 6. The process ofclaim 2, wherein the crowding agent is present in the mixture at aconcentration between 1 to 12% by weight or by volume of the mixture. 7.The process of claim 1, wherein the recombinase comprises a RecA or UvsXprotein.
 8. The process of claim 1, wherein the single-stranded DNAbinding protein (SSB) comprises a prokaryotic SSB protein or amyoviridae gp32 protein.
 9. The process of claim 1, wherein at least oneof the one or more oligonucleotides comprises a detectable label. 10.The process of claim 1, wherein the particles comprise one or more ofthe recombinase, the single-strand binding protein, and at least one ofthe one or more oligonucleotides.
 11. The process of claim 1, whereinthe mixture further comprises one or more of: a recombinase loadingprotein, a reducing agent, creatine kinase, a nuclease, a nucleic acidprobe, and a reverse transcriptase.
 12. The process of claim 1, whereinthe particles are about 1-10 μm in size.
 13. The process of claim 1,wherein detecting particles in the mixture comprises the use ofmicroscopy.
 14. The process of claim 1, wherein detecting particles inthe mixture comprises the use of a lab-on-a-chip device.
 15. The processof claim 1, wherein detecting particles in the mixture comprises the useof flow cytometry.
 16. The process of claim 1, wherein the particles areabout 0.5-20 μm in size.
 17. The process of claim 1, wherein theparticles are detected using fluorescence from the particles.
 18. Theprocess of claim 1, wherein the particles are detected without usingfluorescence from the particles.
 19. The process of claim 1, wherein theparticles comprise a first subset of particles and a second subset ofparticles, the first subset of particles is detected using fluorescencefrom the first subset of particles, and the second subset of particlesare-detected without using fluorescence from the second subset ofparticles.